1. Overview
Cy (Cyanine) series, also called cyanine dyes, namely anthocyanin fluorescent dyes belong to a class of synthetic fluorescent dyes with poly methyl chain chemical structure characteristics. The hypomethyl chain (1-7 hypomethyl) of Cy dyes is often attached to two nitrogen atoms at both ends, one of which is positively charged, thus Cy dyes form a dielectric ionic compound with delocalization of positive charge. Because of this structure, Cy dyes have a high extinction coefficient. The length of the bridge chain and the chromophores at both ends directly control the absorption and emission peaks of the dyes, allowing the Cy series to cover almost all common fluorescence spectrum bands from UV to far infrared. Cyanine dyes are usually divided into fat-soluble and water-soluble Cy dyes.
Cyanine dye is derived from cyanine, which is where its name derives from: Cy2, Cy3, Cy5, and Cy7. All of them can be bonded to nucleic acids, peptides, and proteins by their reaction groups. Cy3 and Cy5 are dyes with high extinction coefficient, which are especially suitable for intracellular sensitive peptide localization experiments and are also the commonly used peptide labeling dyes. Cy5 is sensitive to its surrounding electronic environment, which can be used for enzyme determination. Conformational changes of the attached proteins can lead to positive or negative changes in fluorescence emission. In addition, Cy3 and Cy5 can be used for FRET tests. Cyanine dyes have the highest molar absorption coefficient among fluorescent dyes. The absorption of Cy7 NHS ester, Cy5.5 amine, and Cy3 azide is very low in the background of the near infrared region. Cyanine dyes are also the most stable long-wavelength dyes with the highest fluorescence intensity. They are particularly suitable for small animals in vivo imaginginstead of radioactive elements.
2. Fat-soluble Cy dye
Fat-soluble Cy dyes include Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5. The chromophore of Cy3, Cy5, and Cy7 is indolenine, while that of Cy3.5, Cy5.5, and Cy7.5 is benzoindole. Benzoindole only has one more benzene ring than indolenine. This extra benzene ring makes the absorption and emission peaks of the whole dye red shift so that Cy dye series can cover a wide range of fluorescence spectra. The structure of Cy dye is almost symmetrical. And a 6-carbon chain carboxyl group is attached to one of the chromophores to activate the labeling.
Although it has a positive charge, fat-soluble Cy dye is less water-soluble. When labeling biomolecules (usually in buffer solution), it is often necessary to add organic solution (usually 5-20% DMF or DMSO) to help dissolve. The conventional method is to dissolve the dye in the organic solvent first, and then add it to the aqueous solution of biological molecules in proportion for reaction. After the reaction is complete, the precipitated dye is removed by centrifugation. Of course, fat-soluble Cy dye can also react directly with small organic molecules in organic solvents to label small molecules or polymer materials. Fat-soluble Cy dye is also soluble in chloroform, methanol, THF, acetonitrile, and other conventional organic solvents.
3. Water-soluble Cy dye
By adding sulfonic acid groups to the chromophores of fat-soluble Cy dyes, their water solubility is greatly increased, and their optical stability and quantum yield are slightly improved. Therefore, water-soluble dyes are more tolerant to light and emit slightly stronger fluorescence.
Due to the sulfonic acid, the water solubility of these dyes is very high, and there is no need to add any organic solvent to help dissolve in the labeling reaction. In addition, the labeled dye molecules will not aggregate due to their good water solubility, and will not affect the stability of labeled macromolecules. This is particularly important when multiple dye molecules need to be labeled on each target biomacromolecule, or when the target biomacromolecule has low solubility and poor stability.
4. Cy3 (Cyanine 3)
It is an anthocyanin fluorescent dye appearing orange. The excitation peak and emission peak of Cy3 dye are about 550 nm and 570 nm respectively. Its fluorescence is bright to the naked eye and is pH insensitive. It can be excited by a laser beam at 532nm (acromion) or 556nm (peak) in confocal microscopy. In the ordinary fluorescence microscope, it can be observed by TRITC (tetramethylrhodamine) filter, so it can be used in most fluorescence instruments.
5. Cy7 (Sulfo-Cyanine7)
Cy7 is a fluorescent labeling reagent (Ex=750 nm, Em=773 nm). Cy7 (Cyanine 7) is a near infrared anthocyanin fluorescent dye. Sulfo-Cy7 is its water-soluble form, often collectively referred to as Cy7. Since its fluorescence wavelength (em: ~770 nm) happens to be in the area of near infrared window I of the body (the background fluorescence in this area is weak and long wavelength photons have strong penetration), Cy7 is often used for in vivo imaging of small animals. Cy7 fluorescence is not visible to the naked eye, so it needs to be displayed in imaging software with the help of a CCD camera when viewed under a microscopic lens. In addition, near infrared filters are rare, and many fluorescence microscopes (including confocal microscopes) are not equipped with them. But flow cytometry is often equipped with Cy7 filters, IVIS, and other small animal imaging systems. Cy7 is a commonly used fluorescent dye that can be used to label nucleic acids, proteins, antibodies, peptides, nanoparticles, etc.
Common Cy7 has a low water solubility, so it is necessary to use organic co-solvents in the aqueous phase labeling reaction system. Common organic solvents include DMF, DMSO, and acetonitrile. If the molecules to be labeled are sensitive to organic solvents, sulfonated Cy7 may be selected. The spectral properties of the two are almost identical, so most applications are interchangeable. Sulfonated Cy7 has good water solubility and can be directly used in an aqueous phase labeling reaction system. It does not require organic solvents to help dissolve and is suitable for labeling biomolecules sensitive to organic solvents. In addition, due to the charge of the strongly water-soluble dye molecules themselves, sulfonated Cy7 labeled on the surface of biomolecules will not cause hydrophobic aggregation and affect the stability of the fluorescence-labeled products.
6. Conclusion
Fluorescent labeling of polypeptides is now widely used in biological research since it is not radioactive and easy to be operated. Fluorescent-labeled peptides are used to detect the activity of target proteins, and the high-throughput screening methods developed based on them greatly contribute to drug screening and drug development. BOC Sciences provides varieties of fluorescent-labeled products and services to assist research in related fields.
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